Evans Jason T, Parveen Abida, Smith Grace E, Xu Li, Chan Edward W C, Chan Raphael C Y, Hawkey Peter M
HPA West Midlands Laboratory, Heart of England NHS Foundation Trust, Bordesley Green East, Birmingham B9 5SS, UK.
J Antimicrob Chemother. 2009 Feb;63(2):295-301. doi: 10.1093/jac/dkn506. Epub 2008 Dec 18.
Since the emergence of multidrug-resistant and extensively drug-resistant tuberculosis, there has been a call for a rapid assay to detect rifampicin-resistant strains that can be implemented into a routine service to analyse all strains in a specific geographical location. Denaturing HPLC (dHPLC) is a rapid screening test that can detect mutations in PCR amplicons. The aim of this study was to evaluate the dHPLC analysis of rifampicin-resistant Mycobacterium tuberculosis isolates using an extensive strain collection from Hong Kong and the UK and a collection of 84 consecutive clinical isolates.
DNA from 51 rifampicin-resistant M. tuberculosis strains from the UK and Hong Kong identified from 1996 to 2005 was extracted and each mutation was defined by capillary electrophoresis. A 400 bp PCR product was amplified from each strain, heteroduplexed with a known susceptible control (H37Rv) and analysed by dHPLC at 67.0 degrees C.
Forty-five out of 51 (88.2%) rifampicin-resistant strains with known DNA mutations were detected by dHPLC. Two out of 84 clinical isolates were phenotypically rifampicin-resistant and dHPLC detected a mutation in the rpoB amplicon for both these isolates. dHPLC detected a mutation in 1 out of 82 phenotypically rifampicin-susceptible isolates (M482T, a non-cluster I/II mutation). In a combined analysis of all strains and isolates, mutation detection by dHPLC analysis exhibited 88.2% sensitivity and 98.8% specificity.
This study shows that dHPLC analysis is sensitive and specific and could be implemented in a routine clinical service.
自从多重耐药和广泛耐药结核病出现以来，人们一直呼吁有一种快速检测方法来检测耐利福平菌株，以便能够应用于常规检测服务，对特定地理位置的所有菌株进行分析。变性高效液相色谱法（dHPLC）是一种能够检测PCR扩增子中突变的快速筛查试验。本研究的目的是利用来自中国香港和英国的大量菌株以及84株连续的临床分离株，评估dHPLC对耐利福平结核分枝杆菌分离株的分析。
提取1996年至2005年期间从英国和中国香港鉴定出的51株耐利福平结核分枝杆菌菌株的DNA，并通过毛细管电泳确定每个突变。从每个菌株中扩增出一个400bp的PCR产物，与已知的敏感对照（H37Rv）进行异源双链化，并在67.0℃下通过dHPLC进行分析。
在51株已知DNA突变的耐利福平菌株中，有45株（88.2%）通过dHPLC检测到。84株临床分离株中有2株表型耐利福平，dHPLC检测到这两株分离株的rpoB扩增子中均有突变。dHPLC在82株表型对利福平敏感的分离株中检测到1株有突变（M482T，一种非簇I/II突变）。在对所有菌株和分离株的综合分析中，dHPLC分析检测突变的灵敏度为88.2%，特异性为98.8%。
本研究表明，dHPLC分析具有敏感性和特异性，可应用于常规临床检测服务。
